title: Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular β-galactosidase from Geobacillus stearothermophilus.
abstract: Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a multi-enzyme system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo-acting extracellular enzymes that break down the high-molecular-weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure-function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P2₁2₁2₁, with average crystallographic unit-cell parameters of a=71.84, b=181.35, c=196.57 Å. Full diffraction data sets to 2.45 and 2.50 Å resolution have been collected for both the wild-type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash-cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three-dimensional crystal structure determination of GanB.
authors.affiliation: Institute of Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Hodaya V
authors.initials: HV
authors.lastname: Solomon
authors.forename: Orly
authors.initials: O
authors.lastname: Tabachnikov
authors.forename: Hadar
authors.initials: H
authors.lastname: Feinberg
authors.forename: Lata
authors.initials: L
authors.lastname: Govada
authors.forename: Naomi E
authors.initials: NE
authors.lastname: Chayen
authors.forename: Yuval
authors.initials: Y
authors.lastname: Shoham
authors.forename: Gil
authors.initials: G
authors.lastname: Shoham
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D042541:Intracellular Space / Q000201:enzymology*; D050505:Mutant Proteins / Q000737:chemistry; D017356:Synchrotrons; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: D050505:Mutant Proteins; D001616:beta-Galactosidase
keywords: GH42; GanB; Geobacillus stearothermophilus; β-galactosidase